The overall goal of this research is to understand, within the context of pandemic influenza virus development, the nature of human influenza virus infection of swine cells and pigs. Pandemic influenza viruses are serious threats to public health as natural disease agents and as potential bioterrorism weapons. Recent infections of human beings with H5N1, H9N2, and H7N7 viruses demonstrated that avian influenza viruses can directly cross the species barrier. These viruses have not spread efficiently from person-to-person, but their potential for global transmission throughout the human population could be enhanced by acquiring genes from a human virus. Pigs are widely believed to serve as intermediate hosts for such reassortment between avian and human viruses. In this context, the avian-mammalian interspecies barrier has been widely studied, but little is known regarding control of human virus infection of pigs. The applicant has shown that human viruses of different subtypes and phylogenetic lineages are significantly restricted in their ability to infect primary swine respiratory epithelial cells, yet they readily infect primary human respiratory epithelial cells. In contrast, swine-adapted viruses are highly infectious for both cell types. He has also demonstrated a restriction to human virus infection of pigs in vivo, and that exchange of the HA+NA genes (via reverse genetics) between human and swine viruses is sufficient to fully reverse the restriction to human virus infection of swine cells. The hypothesis addressed in this application is that specific gene sequences within the HA (+/- NA) gene(s) control swine cell infectivity through their impact on virus-receptor interactions and/or virus-cell fusion. In Aim 1, HA or NA single gene reassortant viruses, viruses containing swine x human chimeric genes, and ultimately viruses with site-specific amino acid mutations will be created by reverse genetics. Sequence differences will initially be associated with infectivity phenotype using the applicant's unique primary respiratory epithelial cell culture system. Subsequently, the infectivity of selected viruses will also be assessed in vivo in pigs. In Aim 2, viruses will be compared in their abilities to bind to alpha2,6 vs. alpha2,3 and NAc vs. NGc receptor analogs (solid-phase binding and competitive-inhibition assays), their abilities to fuse with swine vs. human cells (NH4CI resistance and fluorimetric assays), and their sialidase activities (fluorimetric and chromatographic assays). The information gained from this research will substantially enhance the understanding of how pandemic influenza viruses may arise in pigs, and will aid in the development of predictive models of the relative threat posed by novel influenza viruses isolated in the future.